Diagnostic tests are a critical component of an effective strategy to detect and treat biothreat agents. But pathogen evolution or engineering of biothreat pathogens can result in structures that are no longer recognized by diagnostic assays. Next generation biothreat diagnostics will need to permit rapid and flexible assay development to counteract changes that evade diagnostic detection. In this proposal we describe collaborative investigations to develop materials and protocols for flexible and rapid development of multiplex protein detection assays. As a test of these materials and procedures, we will develop a prototype multiplex diagnostic assay to detect and discriminate strains of Ebola virus (EBOV). This development utilizes innovative recombinant systems to develop suitable EBOV antigen, and engineered antibodies, and relies on screening and selection to both shape antibody affinity and to minimize the likelihood of nonspecific interaction when developing multiplex assays. In this proposal we will also explore the development of stable fluorescent reporters and new optical systems to significantly boost the sensitivity of multiplex detection. The system will be tested by simulating an expansion of the multiplex test and testing the effects of our screening concepts. The final optimized assay will be ready for regulated diagnostic development or will be flexible enough to accept new analyte assays the meet clinical and market demands. PUBLIC HEALTH RELEVANCE: Rapid diagnostic tests are essential to a comprehensive strategy to combat biothreat agents. Our work will investigate the design of flexible, sensitive and rapid assays to detect pathogens that have evolved or have been engineered to escape diagnostic detection.